CN119604496A

CN119604496A - ROCK2 inhibitors

Info

Publication number: CN119604496A
Application number: CN202380050515.XA
Authority: CN
Inventors: S·D·瓦克萨尔; A·扎宁-佐洛夫; G·卡奇科夫斯基; M·索卢奇; A·杰德泽扎克; M·克尔瓦纳; D·多玛加拉
Original assignee: Greweton Biotechnology Co ltd
Current assignee: Greweton Biotechnology Co ltd
Priority date: 2022-04-29
Filing date: 2023-04-29
Publication date: 2025-03-11
Also published as: AU2023262720A1; WO2023209692A1; KR20250019030A; EP4514466A1; IL316616A

Abstract

The present disclosure relates to inhibitors of Rho-associated protein kinase (ROCK), pharmaceutical compositions containing such inhibitors, and uses thereof for preventing or treating diseases mediated by such ROCK. In particular, the inhibitors of ROCK are selective for inhibition of ROCK 2.

Description

ROCK2 inhibitors

Technical Field

Background

Rho-associated coiled coil kinase (ROCK) is a serine/threonine kinase from the AGC (PKA, PKG, and PKC) kinase family, and includes two isoforms, ROCK1 and ROCK2. These two isoforms are expressed and regulated differently in specific tissues. For example, ROCK1 is ubiquitously expressed at relatively high levels, while ROCK2 is preferentially expressed in certain tissues, including heart, brain and skeletal muscle. ROCK is a target of small gtpase Rho and is involved in a variety of cellular activities achieved by phosphorylating downstream effector proteins (MLC, LIMK, ERM, MARCKS, CRMP-2, etc.). Studies have shown that various diseases (e.g., pulmonary fibrosis, cardiovascular and cerebrovascular diseases, neurological diseases, cancer, etc.) are associated with ROCK-mediated pathways. Therefore, ROCK has been considered as an important target in the development of new drugs.

Disclosure of Invention

In one aspect, the present disclosure provides a ROCK2 inhibitor having formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is selected from the group consisting of H, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, halo, -CN, C ₁-C₃ perfluoroalkyl, -OR ¹¹、-O-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹;

R ² is selected from the group consisting of H, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, halo, -CN, C ₁-C₃ perfluoroalkyl, -OR ¹¹、-O-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹;

Alternatively, R ¹ and R ² together form a5 OR 6 membered saturated OR unsaturated fused ring, which can contain 0 to 2 ring heteroatoms selected from the group consisting of N, O and S, and which is unsubstituted OR substituted with 1 to 3 substituents selected from the group consisting of C ₁-C₆ alkyl, halo, -CN, -OH, oxo, -O- (C ₁-C₆ alkyl), -O- (C ₁-C₆ alkyl) -OH, -O- (C ₁-C₆ alkyl) -O- (C ₁-C₆ alkyl), -NR ¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、C₁-C₃ perfluoroalkyl, -NR ¹¹-(C₁-C₆ alkyl) NR ¹¹R¹² and-NR ¹¹-(C₁-C₆ alkyl) -OR ¹¹;

x ⁴ is N or CH;

R ³ and R ⁴ are each independently selected from the group consisting of H, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, 3 to 10 membered heterocyclyl, C ₆-C₁₀ aryl, 5 to 14 membered heteroaryl, C _6-12 aralkyl, - (C ₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹ and- (C ₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²;

Alternatively, R ³ and R ⁴ together with the nitrogen to which they are attached provide (i) a 4 to 6 membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from N, O and S, OR (ii) a 5 to 10 membered heterobicyclic ring system having 0 to 3 additional ring heteroatoms selected from N, O and S, wherein the heterocyclic ring OR the heterobicyclic ring system is unsubstituted OR substituted with 1 to 4 substituents selected from the group consisting of halo, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, halo, -CN, C ₁-C₃ perfluoroalkyl, -OR ¹¹, oxo, -O- (C ₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁

-C ₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹.

The dashed line represents an optional double bond;

Each R ⁵ is independently selected from the group consisting of H, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, halo, -CN, C ₁-C₃ perfluoroalkyl, oxo, -OR ¹¹、-O-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹;

n is 0 to 3;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

Each R ⁶ is independently selected from the group consisting of H, C ₁-C₆, alkyl, C ₂-C₆, alkenyl, C ₂-C₆, Alkynyl, C ₃-C₇, cycloalkyl, halo, -CN, C ₁-C₃, perfluoroalkyl, -OR ¹¹、-O-(C₁-C₆, alkyl) -OR ¹¹、-(C₁-C₆, alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆, alkyl) -NR ¹¹R¹²、-(C₁-C₆, alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆, alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆, Alkyl) -OR ¹¹、-(C₁-C₆, alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆, alkyl) _x-C(═O)R¹¹、-(C₁-C₆, alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆, alkyl) _x-C(═O)NR¹¹R¹²、-NR¹¹-(C₁-C₆, Alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆, alkyl) _x-C(═O)OR¹¹;

Each R ⁷ is independently selected from the group consisting of H, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, - (C ₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹ and- (C ₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²;

each x is independently selected from 0 and 1, and

Each R ¹¹ and R ¹² is independently selected from the group consisting of H and C ₁-C₆ alkyl;

Or alternatively, when R ¹¹ and R ¹² are both attached to the same nitrogen, they together form a 4 to 7 membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from the group consisting of N, O and S, and the heterocyclic ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of halo, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, -CN, -NH ₂、C₁-C₃ perfluoroalkyl, -OH, -O- (C ₁-C₆ alkyl) and- (C ₁-C₆ alkyl) -OH.

In another aspect, the present disclosure provides a method for treating a disease or disorder mediated by ROCK2, wherein the method comprises administering to a subject in need thereof an effective amount of a ROCK2 inhibitor provided herein or a pharmaceutically acceptable salt thereof.

In embodiments provided by the present disclosure, the methods are for treating a disease or disorder selected from the group consisting of a fibrotic disease, an inflammatory disease, an autoimmune disease, a cardiovascular disorder, a central nervous system disorder, a neoplastic disease, metabolic syndrome, an ocular disease, a kidney disease, a lung disease, a muscular dystrophy, a sickle cell disease, and a viral disease.

Drawings

Fig. 1 illustrates the structure and characteristics of a compound according to the present disclosure.

FIGS. 2A-2B show that the compounds of example 1 (2A) and example 2 (2B) inhibit IL-17 in human CD4+ T cells stimulated with Th 17-bias conditions.

Figure 3 shows that the compounds of example 1 and example 2 down-regulate STAT3 phosphorylation induced by Th 17-offset activation in human cd4+ T cells.

Figure 4 shows that the compounds of example 1 and example 2 down-regulate pCofilin in human cd4+ T cells.

FIG. 5 shows that the compounds of example 1 and example 2 reduce the expression of a pro-fibrogenic gene in human lung fibroblast MRC-5 cells.

FIG. 6 shows that the compounds of example 1 and example 2 down-regulate collagen type 1 secretion in human lung fibroblast MRC-5 cells.

Fig. 7 shows that the compounds of example 1 and example 2 down-regulate adipogenesis in human adipocytes.

Detailed Description

The invention will now be further described. In the following paragraphs, various aspects of the invention are provided. Each aspect presented may be combined with any other aspect unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The compounds, compositions and methods described herein provide selective inhibitors of Rho-associated coiled coil kinase 2 (ROCK 2) for use in the treatment of diseases or disorders including fibrotic diseases, inflammatory diseases, autoimmune diseases, cardiovascular disorders, central nervous system disorders, neoplastic diseases, metabolic syndrome, ocular diseases, kidney diseases, pulmonary diseases, muscular dystrophy, sickle cell diseases and viral diseases.

The compounds used in the methods and compositions disclosed herein are ROCK inhibitors, particularly ROCK2 selective inhibitors. The compound provides excellent inhibitory activity against ROCK (preferably ROCK 2) and good selectivity (higher selectivity for ROCK2 compared to ROCK 1). The compounds may also provide one or more of good physicochemical properties (e.g., solubility, physical and/or chemical stability), improved pharmacokinetic properties (e.g., improved bioavailability, proper half-life and duration of action), and improved safety (low toxicity and/or fewer side effects, broad therapeutic window). In particular, the ROCK2 inhibitors provided herein may exhibit improved solubility and/or improved bioavailability when administered orally.

or a pharmaceutically acceptable salt thereof, wherein:

x ⁴ is N or CH;

The dashed line represents an optional double bond;

n is 0 to 3;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

each x is independently selected from 0 and 1, and

In some embodiments, the present disclosure provides a ROCK2 inhibitor having formula II:

or a pharmaceutically acceptable salt thereof, wherein:

The dashed line represents an optional double bond;

n is 0 to 3;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

each x is independently selected from 0 and 1, and

In some embodiments, the present disclosure provides a ROCK2 inhibitor having formula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

The dashed line represents an optional double bond;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

each x is independently selected from 0 and 1, and

In some embodiments, the present disclosure provides a ROCK2 inhibitor having formula III:

or a pharmaceutically acceptable salt thereof, wherein:

Alternatively, R ³ and R ⁴ together with the nitrogen to which they are attached provide (i) a 4-to 6-membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from N, O and S, or (ii) a 5-to 10-membered heterobicyclic ring system having 0 to 3 additional ring heteroatoms selected from N, O and S, wherein the heterocyclic ring or the heterobicyclic ring system is unsubstituted or substituted with 1 to 4 substituents selected from the group consisting of halo, C ₁-C₆ alkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, halo, -CN, C ₁-C₃ perfluoroalkyl, -OR ¹¹, oxo, -O- (C ₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) -OR ¹¹、-NR¹¹R¹²、-O-(C₁-C₆ alkyl) -NR ¹¹R¹²、-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)NR¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹;

the dashed line represents an optional double bond;

n is 0 to 3;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

each x is independently selected from 0 and 1, and

In some embodiments, the present disclosure provides a ROCK2 inhibitor having formula IIIa:

or a pharmaceutically acceptable salt thereof, wherein:

-C ₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) -OR ¹¹、-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-O-(C₁-C₆ alkyl) _x-C(═O)R¹¹、-(C₁-C₆ alkyl) _x-C(═O)OR¹¹、-C(═O)-R¹¹、-C(═O)OR¹¹、-(C₁-C₆ alkyl) _x-C(═O)NR ¹¹R¹²、-NR¹¹-(C₁-C₆ alkyl) _x-C(═O)R¹¹ and-NR ¹¹-(C₁-C₆ alkyl) _x-C(═O)OR¹¹.

The dashed line represents an optional double bond;

X ¹ is selected from the group consisting of CR ⁶ and N;

X ² is selected from the group consisting of CHR ⁶、NR⁷, O and S;

X ³ is selected from the group consisting of C, CH and N;

each x is independently selected from 0 and 1, and

For each of the formulae of ROCK2 inhibitors provided herein, in part:

the dashed circle represents one or more optional double bonds. Thus, the partial structure may be unsaturated, have a single double bond at any chemically permissible position, have two double bonds at chemically permissible positions, or be an aromatic ring system. As will be appreciated by those skilled in the art, the double bonds in such ring systems will not be in directly adjacent positions (i.e., they do not share a carbon atom). In an embodiment, the partial structure comprises:

In other embodiments, the portion of the structure comprises:

the term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

The term "halogen" or "halo" means-F, -Cl, -Br or-I. Preferred halogens are-F, -Cl and-Br.

The term "hydroxy" means-OH.

The term "oxo" as used herein refers to an oxygen atom having a double bond to another atom, particularly carbon (i.e., substituent = O).

The term "alkyl" refers to a radical of a saturated aliphatic group, including straight chain alkyl groups and branched chain alkyl groups. Thus, C ₁-C₆ alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and the like.

The term "cycloalkyl" refers to a saturated carbocyclic group having 3 to 7 carbons in the ring. Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "alkenyl" refers to a straight or branched hydrocarbon group having a double bond and 2 to 6 carbon atoms ("C ₂-C₆ alkenyl"). Alkenyl includes ethenyl, 1-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl and the like. When a compound of the present disclosure contains an alkenyl group, the compound may exist as an E-form, a Z-form, or any mixture thereof.

The term "alkynyl" refers to a straight or branched hydrocarbon radical ("C ₂-C₆ alkynyl") having a triple bond and 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, and the like.

The term "aryl" as used herein includes 5-and 6-membered monocyclic aromatic groups which may include zero to four heteroatoms, such as benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles", "heteroaromatics" or "heteroaryl groups". The term "aryl" also includes 7-to 14-membered polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (rings are "fused rings"), wherein at least one of the rings is aromatic (including heteroaryl), e.g., the other rings may be fused cycloalkyl, cycloalkenyl, aryl, heteroaryl, and/or heterocyclyl. The monocyclic heteroaryl group may have 1 to 3 ring heteroatoms and the fused polycyclic heteroaryl group may have 1 to 5 ring heteroatoms, wherein the ring heteroatoms are selected from N, O and S.

The term "heterocyclyl" or "heterocyclic group" refers to a 3-to 10-membered ring structure, more preferably a 5-or 6-membered ring, the ring structure of which comprises one to four heteroatoms. The heterocycle may also be polycyclic. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenoxazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxacyclopentane, thiacyclopentane, oxazole, piperidine, piperazine, morpholine, lactone, lactams such as azetidinone and pyrrolidone, sultam, sultone, and the like.

The term "aralkyl" as used herein refers to a C ₁-C₆ alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

As used herein, when each expression (e.g., alkyl, m, n, R, etc.) occurs more than one time in any structure, its definition is intended to be independent of its definition elsewhere in the same structure.

It is to be understood that "substituted", "substituted" or "substituted" includes the proviso that such substitution is in accordance with the permissible valence of the substitution atoms and substituents and that the substitution results in a stable compound that does not undergo conversion by itself, such as by rearrangement, cyclization, elimination, and the like, for example.

Certain compounds provided in the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis-and trans-isomers, R-and S-enantiomers, diastereomers, racemic mixtures thereof, and other mixtures thereof, falling within the scope of the invention. Other asymmetric carbon atoms may be present in the substituents, such as alkyl groups. All such isomers and mixtures thereof are included in the present invention.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds disclosed herein as well as the inorganic and organic base addition salts of the compounds disclosed herein.

As described above, certain embodiments of ROCK2 inhibitors may contain basic functional groups, such as amino groups, and are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. These salts may be prepared in situ in the administration vehicle or in the dosage form manufacturing process, or by separately reacting the purified compounds of the invention in free base form with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthalate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like. (see, e.g., berge et al (1977) "Pharmaceutical Salts", J.Pharm. Sci.66:1-19).

Pharmaceutically acceptable salts of the compounds of the invention include the conventional non-toxic salts or the venenum salts of the compounds, for example from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic (isothionic), and the like.

In other cases, the compounds provided in the present disclosure may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can likewise be prepared in situ in the administration vehicle or in the dosage form manufacturing process, or by separately reacting the purified compound in the free acid form with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with aqueous ammonia, or with a pharmaceutically acceptable primary, secondary or tertiary organic amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, aluminum salts, and the like. Representative organic amines useful in forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (see, e.g., berge et al, supra).

ROCK2 inhibitors provided herein may exhibit improved solubility and/or improved bioavailability when administered orally. For example, the compounds of examples 1 and 2 showed kinetic solubilities of 2.2 μg/ml and 2.69 μg/ml, respectively, which was significantly improved compared to comparative compound a having kinetic solubilities <1.9 μg/ml.

Therapeutic method

The present disclosure provides methods for preventing or treating a disease mediated by ROCK2, wherein the methods comprise administering to a subject in need thereof an effective amount of a ROCK2 inhibitor as disclosed herein or a pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure provides a method for treating at least one disease or disorder selected from the group consisting of a fibrotic disease, an inflammatory disease, and an autoimmune disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound or composition as defined herein.

In other embodiments, the present disclosure provides a method for treating a cardiovascular disorder, a central nervous system disorder, a neoplastic disease, or metabolic syndrome, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound or composition as defined herein.

In some embodiments, the disease mediated by ROCK2 is an autoimmune disorder, including rheumatoid arthritis, systemic lupus erythematosus (SLE; lupus), psoriasis, psoriatic arthritis, multiple sclerosis, crohn's disease, ulcerative colitis, atopic dermatitis, eczema, or graft versus host disease (GVHD; acute and chronic), idiopathic pulmonary fibrosis, and scleroderma.

Other autoimmune disorders that may be treated according to the methods provided in the present disclosure include Acute Disseminated Encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, edison's disease, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, anti-phospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune Inner Ear Disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune, Autoimmune retinopathy, autoimmune thyroid disease, autoimmune urticaria, axon & neuronal neuropathy, baluo disease, white race disease, bullous pemphigoid, cardiomyopathy, castelman's disease, celiac disease, chagas' disease, chronic inflammatory demyelinating peripheral neuropathy (CIDP), and chronic recurrent multifocal encephalomyelitis (CRMO), chager-Schmitt syndrome, kegen syndrome, coxsackie viral myocarditis, CREST disease, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, devic disease (neuromyelitis optica), discoid lupus, deretsler syndrome, eosinophilic esophagitis, Eosinophilic fasciitis, sarcoidosis, EWens syndrome, fibroalveolar inflammation, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, goldpasm's syndrome, granulomatous Polyangiitis (GPA), graves ' disease, guillain-Barre syndrome, behcet's encephalitis, hashimoto thyroiditis, hemolytic anemia, glomerulonephritis, hypogammaglobulinemia, idiopathic Thrombocytopenic Purpura (ITP), igA nephropathy, igG 4-related sclerotic diseases, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile myositis, kawasaki syndrome, Lambert-eaton syndrome, white blood cell disruption vasculitis, lichen planus, lichen sclerosus, wood-like conjunctivitis, linear IgA disease (LAD), meniere's disease, microscopic polyangiitis, mixed Connective Tissue Disease (MCTD), mo Lunshi ulcers, murrah-Haydig disease, myasthenia gravis, myositis, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, recurrent rheumatism, pediatric Autoimmune Neuropsychiatric Disorder (PANDAS) with streptococcal infection, paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria (PNH), para-Luo Ershi syndrome, Pason-tenna syndrome, pars plana (external Zhou Putao membranitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyadendric syndrome (type I, II, III), polymyalgia rheumatica, polymyositis, post myocardial infarction syndrome, post pericardial osteotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriatic arthritis, pustular gangrene, pure red cell regeneration disorder, reynolds phenomenon, reactive arthritis, reflex inductive dystrophy, leiter's syndrome recurrent polyarthritis, Retroperitoneal fibrosis, sarcoidosis, schmidt syndrome, scleritis, sjogren's syndrome, sperm and testis autoimmunity, stiff person syndrome, subacute Bacterial Endocarditis (SBE), susac syndrome, sympathogenic ophthalmia, tawnian arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), tole-hentwo syndrome, transverse myelitis, undifferentiated Connective Tissue Disease (UCTD), autoimmune diabetes type 1, uveitis, vasculitis, vesicular dermatosis, vitiligo and wegener granulomatosis (granulomatous polyangiitis; GPA).

Inflammatory conditions that may be treated by the methods provided in the present disclosure include, but are not limited to, cardiovascular inflammation, pulmonary inflammation, renal inflammation, arteriosclerosis, and sepsis.

Fibrotic conditions treatable by the methods provided in the present disclosure include idiopathic pulmonary fibrosis (idiopathic pulmonary fibrosis), renal fibrosis (renal fibrosis), ocular fibrosis, cardiac fibrosis, NASH, scleroderma, systemic sclerosis, and cirrhosis.

In another embodiment, the present disclosure provides a method for treating muscular dystrophy (duchenne muscular dystrophy). In another embodiment, the present disclosure provides a method for treating myotonic muscular dystrophy.

In other embodiments, the ROCK2 inhibitors provided herein are useful for inhibiting tumor cell growth and metastasis and angiogenesis, and for treating neoplastic diseases. Neoplastic diseases include any malignant growth or tumor caused by abnormal or uncontrolled cell division. Neoplastic diseases include lymphomas, carcinomas, leukemias, sarcomas, and blastomas. Non-limiting examples include squamous cell carcinoma, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic tumor, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

In other embodiments, the ROCK2 inhibitors provided herein are useful in the treatment of cardiovascular disorders, including hypertension, cardiomyopathy, cardiac remodeling, atherosclerosis, restenosis, cardiac hypertrophy, cerebral ischemia, cerebral vasospasm, and erectile dysfunction.

In other embodiments, the ROCK2 inhibitors provided herein are useful in the treatment of pulmonary disorders, including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and asthma.

In other embodiments, the ROCK2 inhibitors provided herein are useful in the treatment of central nervous system disorders, including neuronal degeneration or spinal cord injury, traumatic brain injury, cerebral spongiform vascular malformations, huntington's disease, parkinson's disease, alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), or multiple sclerosis.

In other embodiments, the present disclosure provides methods for treating kidney disease, including polycystic kidney disease, kidney fibrosis, and diabetic kidney disease.

In other embodiments, the ROCK2 inhibitors provided herein are useful in the treatment of metabolic diseases, including insulin resistance, hyperinsulinemia, type 2 diabetes, obesity, metabolic syndrome, and glucose intolerance. ROCK2 inhibitors may be used to achieve weight loss and/or limit weight gain. In one embodiment, the ROCK2 inhibitor is used to reduce or prevent insulin resistance or restore insulin sensitivity.

In other embodiments, the ROCK2 inhibitors provided herein are useful in the treatment of ocular disorders, including ocular hypertension, age-related macular degeneration (AMD; wet and dry), choroidal Neovascularization (CNV), choroidal tumors, diabetic Macular Edema (DME), iris neovascularization, uveitis, glaucoma, primary open angle glaucoma, acute angle closure glaucoma, pigmentary glaucoma, congenital glaucoma, normal tension glaucoma, secondary glaucoma, neovascular glaucoma, geographic atrophy, and retinitis of premature infants (ROP).

In another embodiment, the present disclosure provides a method for treating sickle cell disease.

In other embodiments, the ROCK2 inhibitors provided herein are useful for treating (i.e., curing or lessening the severity of, etc.) viral infections (particularly coronavirus infections such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV) and for treating or preventing sequelae caused by viral infections (including coronavirus infections such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV). In some embodiments, the viral infection is a SARS-CoV-1 infection. In some embodiments, the viral infection is a SARS-CoV-2 infection. In some embodiments, the viral infection is a MERS-CoV infection. In embodiments, the sequelae comprise one or more of the group consisting of fatigue, dyspnea (dyspnea), cough, joint pain (osteo-articular pain), myalgia, headache, chest pain, fever, palpitations, myocardial inflammation, ventricular dysfunction, stroke, pulmonary dysfunction, fibrosis (such as pulmonary fibrosis), renal dysfunction, rash, hair loss, olfactory and/or gustatory dysfunction, sleep dysregulation, altered cognitive impairment, memory impairment, depression, anxiety, mood changes, and combinations thereof. In embodiments, the sequelae include inflammation and/or fibrosis.

Pharmaceutical composition

In one aspect, the present disclosure provides a pharmaceutically acceptable composition for treating a viral disease comprising a therapeutically effective amount of one or more ROCK2 inhibitors provided by the present disclosure formulated with one or more pharmaceutically acceptable carriers. As described below, the pharmaceutical compositions of the present disclosure may be specifically formulated for administration in solid or liquid form, including those suitable for (1) oral administration, e.g., drenching (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes for application to the tongue, (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, as, e.g., sterile solutions or suspensions or sustained release formulations, (3) topical application, e.g., as a cream, ointment or controlled release patch or spray, to the skin, (4) intravaginal or intrarectal administration, e.g., as a suppository, pessary, cream or foam, (5) sublingual administration, (6) ocular administration, (7) transdermal administration, or (8) nasal administration.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium or zinc stearate, or stearic acid), solvent or solvent encapsulating material, which involves carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier should be compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that may be used as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose, (2) starches such as corn starch and potato starch, (3) celluloses and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, (4) tragacanth powder, (5) malt, (6) gelatin, (7) talc, (8) excipients such as cocoa butter and suppository waxes, (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols such as propylene glycol, (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) ringer's solution, (19) ethanol, (20) pH buffer solution, (21) polyesters, polycarbonates and/or polyanhydrides, and (22) other compatible substances used in pharmaceutical formulations.

The compounds of the present disclosure may be formulated with conventional carriers and excipients which may be selected according to conventional practice. The tablets may contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and, when intended for delivery by means other than oral administration, may generally be isotonic. All formulations may optionally contain excipients such as those described in "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methylcellulose, stearic acid, and the like.

Although the ROCK2 inhibitors disclosed herein (referred to herein as "active ingredients") may be administered alone, they are preferably provided as pharmaceutical formulations. Formulations of the present disclosure for both veterinary and human use comprise at least one active ingredient as provided above, together with one or more acceptable carriers for the active ingredient, and optionally other therapeutic ingredients, particularly those additional therapeutic ingredients as discussed herein.

Formulations include those suitable for the route of administration provided herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing co., easton, pa.). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present disclosure suitable for oral administration may be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally be formulated so as to provide slow or controlled release of the active ingredient therein.

For infections of the eye or other external tissues, such as the oral cavity and skin, the formulation is preferably applied as a topical solution, ointment or cream containing the active ingredient. The active ingredient may be present in an amount of, for example, 0.075 to 20% w/w (including active ingredients in increments of 0.1% w/w ranging between 0.1% and 20%, such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w, most preferably 0.5 to 10% w/w. When formulated as ointments, the active ingredients may optionally be used with paraffin or a water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base can include, for example, at least 30% w/w of a polyol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, 1, 3-butanediol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may desirably contain compounds that enhance absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin permeation enhancers include dimethyl sulfoxide and related analogs.

The oil phase of the emulsions of the present disclosure may be composed of known ingredients in a known manner. While this phase may contain only an emulsifier (otherwise known as a diarrhea agent), it desirably contains a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. And also preferably comprises oil and fat. The emulsifying agent, with or without the stabilizing agent, constitutes a so-called emulsifying wax, and the wax, together with the oil and fat, constitutes a so-called emulsifying ointment base which forms the oily dispersed phase of the ointment formulation.

Emulsifying agents and emulsion stabilizers suitable for use in the formulations of the present disclosure include60、80. Cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. Other emulsifiers and emulsion stabilizers suitable for use in the formulations of the present disclosure include80。

The selection of a suitable oil or fat for the formulation is based on achieving the desired characteristics. The cream should preferably be a non-greasy, non-staining and washable product having a consistency suitable to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic alkyl esters may be used, such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or blends of branched chain esters known as Crodamol CAP, the last three being preferred esters. These substances may be used alone or in combination depending on the desired properties. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used.

Pharmaceutical formulations according to the present disclosure comprise a combination according to the present disclosure together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. The pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral administration, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may contain one or more agents, including sweeteners, flavoring agents, coloring agents, and preservatives, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binders such as starch, gelatin or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed, alone or with a wax.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example starch, mannitol, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

The aqueous suspensions of the present disclosure contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth, and gum acacia, and dispersing or wetting agents, such as naturally-occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetyl alcohol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives (such as ethyl or n-propyl parahydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents (such as sucrose or saccharin). Other non-limiting examples of suspensions include cyclodextrin and Captisol (=sulfobutyl ether β -cyclodextrin; SEB- β -CD).

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspension may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners (such as those described above) and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible granules and granules of the present disclosure suitable for preparing an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the present disclosure may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil (such as olive oil or arachis oil), a mineral oil (such as liquid paraffin) or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally-occurring phosphatides, such as soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain a sweetener or flavoring agent. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the present disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. Such suspensions may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1, 3-butanediol, or as a lyophilized powder. Acceptable vehicles and solvents that may be used include water, ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (such as oleic acid) find use in the preparation of injectables. Acceptable vehicles and solvents that may be used are water, ringer's solution, isotonic sodium chloride solution, and hypertonic sodium chloride solution.

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form can vary depending upon the host treated and the particular mode of administration. For example, a timed release formulation intended for oral administration to humans may contain about 1 to 1000mg of the active substance together with a suitable and convenient amount of carrier material, which may constitute from about 5% to about 95% by weight of the total composition. The pharmaceutical compositions may be prepared to provide an easily measurable dosage. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μg of active ingredient per milliliter of solution so that infusion of a suitable volume at a rate of about 30mL/hr may be performed.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient. The active ingredient may be present in such formulations at a concentration of 0.5% to 20%, advantageously 0.5% to 10%, and in particular about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, typically sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis, such as gelatin and glycerin or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as suppositories with a suitable base containing, for example, cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal administration have a particle size, for example, in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 microns, etc., which are administered by rapid inhalation through the nasal passages or by oral inhalation to reach the alveolar vesicles. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds.

Formulations suitable for vaginal administration may be presented as suppositories, pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile granules, granulates and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose or suitable portion thereof of the active ingredient as described above.

The present disclosure also provides a veterinary composition comprising at least one active ingredient as defined above and a veterinary carrier for the active ingredient.

Veterinary carriers are materials used for the purpose of administering the compositions and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary arts and compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

The compounds of the present disclosure are useful for providing controlled release pharmaceutical formulations ("controlled release formulations") containing one or more compounds of the present disclosure as an active ingredient, wherein the release of the active ingredient is controlled and regulated to allow for less frequent administration or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

The patient receiving the treatment is any animal in need thereof, including primates, particularly humans, and other mammals such as horses, cattle, pigs, and sheep, as well as poultry and pets in general.

Examples

Example 1

Method A. Taking the synthesis of 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine as an example.

Step 1 Synthesis of intermediate 1:6-bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1H-indole

6-Bromoindole-2-carboxylic acid (5.26 g,21.91mmol,1.00 eq.) was dissolved in anhydrous DCM. Oxalyl chloride (2.04 mL,24.10mmol,1.10 eq.) was then added dropwise to the reaction mixture (25.0 mL) at 0 ℃ followed by several drops of DMF. The mixture was stirred at 0 ℃ for 1h, then the cold bath was removed and stirring was continued for 3h at room temperature. TLC analysis showed complete conversion of starting material and UPLC-MS analysis showed quality corresponding to the appropriate ester (quenching the sample with MeOH). The reaction mixture was evaporated to dryness and co-evaporated twice with ACN. The resulting cream-colored powder was then redissolved in THF (25.0 mL) and transferred dropwise via syringe to a stirred ice-cold solution of 3, 3-difluoroazetidine hydrochloride (2.84 g,21.91mmol,1.00 eq.) and N, N-diisopropylethylamine (15.27 mL,87.65mmol,4.00 eq.) in DCM (25.0 mL). After 1 hour at 0 ℃, the cold bath was removed and stirred overnight at room temperature. Then NaHCO ₃ solution was added and the mixture of DCM and THF was removed in vacuo. The precipitate was collected by vacuum filtration, washed with water, and dried under high vacuum for 18H to give 6-bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1H-indole (6.5 g,90% yield) ).¹HNMR(400MHz,DMSO-d₆)δ11.93–11.86(m,1H),7.65–7.59(m,2H),7.21(dd,J＝8.6,1.8Hz,1H),6.95(dd,J＝2.2,0.9Hz,1H),4.77(m,J＝170.3Hz,4H).UPLC-MS,m/z:[M+H]＝314.8

Step 2 Synthesis of 6-bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indole.

To a solution of 6-bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1H-indole (1.20 g,3.81mmol,1.00 eq.) in anhydrous DMF (15.0 mL) was added NaH (0.12 g,4.57mmol,1.20 eq.) in portions at 0 ℃. The resulting mixture was stirred at the same temperature for 1h. Methyl iodide (0.26 mL,4.19mmol,1.10 eq.) was then added dropwise. Stirring was continued for 30 minutes at 0 ℃ and then at room temperature overnight. The progress of the reaction was monitored using TLC analysis (eluent 10% MeOH-DCM). The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na ₂SO₄, filtered and concentrated to give 6-bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indole (1.09 g,70% yield), which was used in the next step without purification .¹H NMR(400MHz,DMSO-d₆)δ7.87–7.84(m,1H),7.59(d,J＝8.5Hz,1H),7.25(dd,J＝8.5,1.7Hz,1H),7.04(d,J＝0.8Hz,1H),5.04–4.38(m,4H),3.93(s,3H).UPLC-MS,m/z:[M+H]⁺＝330.75

Step 32- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-6- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-indole (intermediate 1).

6-Bromo-2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indole (0.74 g,2.25mmol,1.00 eq.), bis (pinacolato) diboron (0.85 g,3.37mmol,1.50 eq.) and potassium acetate (0.66 g,6.74mmol,3.00 eq.) are dissolved in anhydrous dioxane (9.0 mL). The reaction mixture was purged 4 times with argon, then 1,1' -bis (diphenylphosphino) -ferrocene-palladium (II) (0.05 g,0.06mmol,0.03 eq.) was added. The reaction mixture was placed in a preheated oil bath at 90 ℃ overnight. The reaction mixture was cooled to room temperature, filtered through a celite pad, concentrated and purified using flash column chromatography (0-10% MeOH in DCM, gradient elution) to give 2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-6- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-indole (intermediate 1) (0.81 g,89% yield ).¹H NMR(400MHz,DMSO-d₆)δ7.83(d,J＝0.9Hz,1H),7.63(dd,J＝8.0,0.8Hz,1H),7.42(dd,J＝8.0,0.9Hz,1H),7.03(d,J＝0.9Hz,1H),4.70(d,J＝105.5Hz,5H),3.98(s,3H),1.33(s,11H).UPLC-MS,m/z:[M+H]⁺＝377.15.

Step 4 Synthesis of intermediate 2:2-chloro-N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine

To a solution of 4- (1H-imidazol-5-yl) aniline (0.89 g,5.60mmol,1.00 eq.) in ethanol (9.0 mL) was added N, N-Diisopropylethylamine (DIPEA) (1.46 mL,8.38mmol,1.50 eq.) and the reaction mixture was stirred at room temperature for 15 min. 2, 4-dichloropyrimidine (0.83 g,5.6mmol,1.00 eq.) was then added and the reaction mixture stirred for 24h. It was filtered. The resulting solid cake was redissolved in boiling ethanol and filtered again and dried under high vacuum to give 2-chloro-N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine (0.89 g,56% yield) ).¹H NMR(300MHz,DMSO-d6)δ12.90(s,1H),10.15(s,1H),8.14(d,J＝5.9Hz,1H),8.03(s,2H),7.60(d,J＝2.4Hz,4H),6.78(d,J＝5.9Hz,1H).UPLC-MS,m/z:[M+H]⁺＝271.95.

Step 5 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine.

A mixture of intermediate 1 (0.20 g,0.49mmol,1.40 eq), intermediate 2 (0.10 g,0.35mmol,1.00 eq) and sodium carbonate (0.07 g,0.70mmol,2.00 eq) was dissolved in a mixture of dimethoxyethane (6.0 mL) and water (2.0 mL) and degassed with argon for 15 minutes. Tetrakis (triphenylphosphine) palladium (0.024 g,0.021mmol,0.06 eq) was then added and the reaction mixture stirred in a microwave reactor at 110 ℃. The progress of the reaction was monitored using UPLC-MS analysis and after stirring for 4 hours the starting material was completely converted. After cooling, the mixture was diluted with EtOAc and filtered through a celite pad. The filtrate was concentrated in vacuo and purified via preparative HPLC (mobile phase: H ₂ o+0.1% FA, acn+0.1% FA column C18 preparation) to afford the compound 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine as a yellow foam, formate salt (0.060g,35％).¹H NMR(400MHz,DMSO-d6)δ9.69(s,1H),8.56(s,1H),8.41(d,J＝5.8Hz,1H),8.28(d,J＝7.2Hz,2H),8.21(dd,J＝8.5,1.4Hz,1H),7.81(s,4H),7.75(d,J＝8.5Hz,1H),7.71(d,J＝1.1Hz,1H),7.55(s,1H),7.08(s,1H),6.73(d,J＝5.8Hz,1H),4.73(d,J＝111.7Hz,4H),4.05(s,3H).LCMS,m/z:[M+H]⁺＝486.41.

Example 2

Method B- -Synthesis of 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine is exemplified.

Step 1': synthesis of intermediate 2':1- (azidomethyl) -4-methoxybenzene

A mixture of 4-methoxybenzyl chloride (2.00 g,12.77mmol,1.00 eq.) and sodium azide (0.99 g,15.32mmol,1.20 eq.) in anhydrous dimethyl sulfoxide (20.0 mL) was stirred overnight at room temperature. The progress of the reaction was monitored by TLC analysis (eluent: 9:1DCM: meOH). The mixture was partitioned between water and EtOAc. The aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na ₂SO₄, filtered and evaporated to dryness to give 1- (azidomethyl) -4-methoxybenzene (1.98 g, 94%) as a pale yellow liquid.

Step 2': synthesis of 4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } aniline

To a mixture of 1- (azidomethyl) -4-methoxybenzene (1.98 g,12.013mmol,1.0 eq.) and 4-ethynylaniline (1.40 g,12.00mmol,1.00 eq.) in anhydrous dimethyl sulfoxide (29.70 mL) was added copper (II) sulfate pentahydrate (0.60 g,2.40mmol,0.20 eq.) followed by sodium ascorbate (0.92 g,4.80mmol,0.40 eq.) and the resulting mixture was stirred at room temperature for 2h. Complete conversion of the starting material was observed according to UPLC-MS analysis. The mixture was poured into cold water. The precipitated solid was filtered, washed with water and dried to give 4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } aniline as a yellow solid (2.5g,73％).¹H NMR(300MHz,DMSO-d6)δ8.26(s,1H),7.48(d,J＝8.0Hz,2H),7.37–7.23(m,2H),7.00–6.87(m,2H),6.58(d,J＝8.0Hz,2H),5.49(s,2H),5.19(s,2H),3.74(s,3H).UPLC-MS,m/z:[M+H]⁺＝281.10.

Step 3': synthesis of 2-chloro-N- (4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine

To a solution of 4- {2- [ (4-methoxyphenyl) methyl ] -2H-1,2, 3-triazol-4-yl } aniline (2.50 g,8.83mmol,1.00 eq.) in ethanol (15.0 mL) was added N, N-diisopropyl-ethylamine (DIPEA) (1.999 mL,11.48mmol,1.30 eq.) and the reaction mixture was stirred at room temperature for 15 min. 2, 4-dichloropyrimidine (1.31 g,8.83mmol,1.00 eq.) was then added and the reaction mixture was stirred overnight at 90 ℃. It was evaporated to dryness. The crude product was purified by FCC (0-5% MeOH in DCM) to give 2-chloro-N- (4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine as a yellow solid (1.43g,40％).¹H NMR(300MHz,DMSO-d6)δ10.13(s,1H),8.52(s,1H),8.18(d,J＝5.9Hz,1H),7.83(d,J＝8.6Hz,2H),7.66(d,J＝8.3Hz,2H),7.34(d,J＝8.4Hz,2H),7.01–6.91(m,2H),6.79(d,J＝5.9Hz,1H),5.56(s,2H),3.74(s,3H).UPLC-MS,m/z:[M+H]^-＝392.10.

Step 4':2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- (4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine.

A mixture of intermediate 1 (0.35 g,0.42mmol,1.10 eq), intermediate 2' (0.25 g,0.38mmol,1.00 eq.) and sodium carbonate (0.12 g,1.14mmol,3.00 eq.) was dissolved in a mixture of dimethoxyethane (6.0 mL) and water (2.0 mL) and degassed with argon for 10 minutes. Then, tetrakis (triphenylphosphine) palladium (0.04 g,0.04mmol,0.10 eq) was added and the reaction mixture stirred in a microwave reactor at 110 ℃. The progress of the reaction was monitored using UPLC-MS analysis and after stirring for 4 hours the starting material was completely converted. After cooling, the mixture was diluted with EtOAc and filtered through a celite pad. The filtrate was concentrated to dryness to give the crude compound 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- (4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine (0.2 g) which was used in the next step without further purification. UPLC-MS, M/z: [ M=H ] ⁺ = 607.10. ¹ H NMR analysis was not performed.

Step 5':2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine.

2- [2- (3, 3-Difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- (4- {2- [ (4-methoxyphenyl) methyl ] -2H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine (0.20 g,0.20mmol,1.00 eq) was dissolved in trifluoroacetic acid (1.06 mL,13.85mmol,70.00 eq) and the reaction mixture was stirred at 100℃overnight. After this, no partial conversion was observed. The reaction mixture was evaporated to dryness and purified using preparative HPLC (mobile phase: ACN+0.1% FA, H2O+0.1% FA, column C18 preparation) to give 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -1-methyl-1H-indol-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine (0.025g,26％).¹H NMR(400MHz,DMSO-d6)δ9.89(s,1H),8.57(s,1H),8.44(d,J＝5.9Hz,1H),8.32(d,J＝16.4Hz,2H),8.21(d,J＝8.6Hz,1H),7.93(q,J＝8.6Hz,4H),7.76(d,J＝8.6Hz,1H),7.09(s,1H),6.78(d,J＝5.8Hz,1H),4.86(s,4H),4.06(s,3H).LCMS,m/z:[M＝H]⁺＝487.18.

Example 3

Method C. Take the synthesis of 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-c ] pyridin-6-yl ] -N- [4- (1H-imidazol-4-yl) phenyl ] pyrimidin-4-amine (example 3) and 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-c ] pyridin-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine (example 3').

Step 1 and step 2 Synthesis of intermediate 1:3, 3-difluoro-1- {4H,5H,6H, 7H-thieno [2,3-c ] pyridine-2-carbonyl } azetidine

6- [ (Tert-Butoxycarbonyl ] -4H,5H,6H, 7H-thieno [2,3-c ] pyridine-2-carboxylic acid (0.20 g,0.70mmol,1.00 eq.) was dissolved in DMF (5 mL), followed by addition of DIPEA (0.50 mL,2.83mmol,4.00 eq.) to the reaction mixture followed by HATU (0.32 g,0.84mmol,1.20 eq.). The reaction mixture was stirred at room temperature for 15 minutes and 3, 3-difluoroazetidine hydrochloride (0.11 g,0.85mmol,1.20 eq.) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched by addition of water and the aqueous layer extracted with EtOAc. The combined organic layers were washed with brine, dried over Na ₂SO₄, filtered and concentrated to give tert-butyl 2- (3, 3-difluoroazetidine-1-carbonyl) -4h,5h,6h,7 h-thieno [2,3-c ] pyridine-6-carboxylate, [ m+acn ] ⁺ = 358.90. The crude material was used in the next step without further purification. To the resulting residue was added 2M HCl diAn alkane solution (2.00 mL,8.20mmol,11.00 eq.) and the mixture was stirred at room temperature for 12h. The solvent was evaporated to dryness and the resulting HCl salt of 3, 3-difluoro-1- {4h,5h,6h,7 h-thieno [2,3-c ] pyridine-2-carbonyl } azetidine (0.25 g) was used directly in the next step. UPLC-MS, M/z: [ M+ACN ] ⁺ = 299.80.

Step 3 Synthesis of 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-c ] pyridin-6-yl ] -N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine.

To a solution of intermediate 1 (0.17 g,0.33mmol,1.00 eq.) in acetonitrile (5.0 mL) was added N, N-Diisopropylethylamine (DIPEA) (0.23 mL,1.33mmol,4.00 eq.) and the reaction mixture was stirred at room temperature for 15 min. Intermediate 2 (0.10 g,0.33mmol,1.00 eq.) was then added and the reaction mixture was stirred at 95 ℃ overnight. A low conversion of the starting material was observed (according to the UPLC-MS analysis). Cs ₂CO₃ (0.21 g,0.66mmol,2.00 eq.) was added to the reaction mixture and stirring was continued for an additional 12hr at the same temperature. The reaction mixture was then quenched by addition of water and extracted with EtOAc. The combined organic layers were dried over Na ₂SO₄, filtered, concentrated and purified by preparative HPLC (mobile phase: ACN+0.1% FA, H2O+0.1% FA column C18 preparation) to afford 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-C ] pyridin-6-yl ] -N- [4- (1H-imidazol-5-yl) phenyl ] pyrimidin-4-amine (0.003g,2％).¹H NMR(400MHz,DMSO-d6)δ9.37(s,1H),8.42(s,5H),7.97(d,J＝5.7Hz,1H),7.67(dd,J＝26.6,10.4Hz,6H),7.51(s,1H),7.34(s,1H),6.11(d,J＝5.7Hz,1H),4.98(s,2H),4.04(t,J＝5.8Hz,3H),2.75(s,3H).LCMS,m/z:[M+H]⁺＝493.85.

Step 4 2- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-c ] pyridin-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine.

2- [2- (3, 3-Difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-C ] pyridin-6-yl ] -N- (4- {1- [ (4-methoxyphenyl) methyl ] -1H-1,2, 3-triazol-4-yl } phenyl) pyrimidin-4-amine (0.20 g,0.20mmol,1.00 eq.) was dissolved in trifluoroacetic acid (1.06 mL,13.85mmol,70.00 eq.) and the reaction mixture was stirred overnight at 100 ℃. After this, no partial conversion was observed. The reaction mixture was evaporated to dryness and purified using preparative HPLC (mobile phase: ACN+0.1% FA, H2O+0.1% FA, column C18 preparation) to give 22- [2- (3, 3-difluoroazetidine-1-carbonyl) -4H,5H,6H, 7H-thieno [2,3-C ] pyridin-6-yl ] -N- [4- (1H-1, 2, 3-triazol-4-yl) phenyl ] pyrimidin-4-amine (0.025g,26％).¹H NMR(400MHz,DMSO-d6)δ9.50(s,1H),8.35(s,1H),8.27(s,1H),8.00(d,J＝5.7Hz,1H),7.83(d,J＝8.7Hz,2H),7.75(d,J＝8.7Hz,2H),7.34(s,1H),6.13(d,J＝5.7Hz,1H),4.99(s,2H),4.67(s,4H),4.05(t,J＝5.7Hz,2H),2.76(d,J＝6.0Hz,2H). formate. LCMS, M/z: [ m+h ] += 495.11.

Example 4

(Method A)

¹H NMR(300MHz,DMSO-d6)δ12.51(s,1H),9.71(s,1H),8.47(s,1H),8.40(d,J＝5.9Hz,1H),8.20(dd,J＝8.4,1.3Hz,1H),8.14(s,1H),7.81(d,J＝2.6Hz,5H),7.75(d,J＝8.4Hz,1H),7.55(s,1H),7.09(s,1H),6.71(d,J＝5.9Hz,1H),5.52(s,2H),4.69(d,J＝89.6Hz,4H),3.18(s,3H),2.86(s,3H). Formate. LCMS, M/z: [ m+h ] ⁺ = 557.33.

Example 5

(Method A)

¹H NMR(400MHz,DMSO-d6)δ9.66(s,1H),8.55(s,1H),8.40(d,J＝5.8Hz,1H),8.19(dd,J＝8.4,1.4Hz,1H),8.16(s,1H),7.80(s,4H),7.73(d,J＝8.4Hz,1H),7.70(d,J＝1.1Hz,1H),7.51(s,1H),7.00(s,1H),6.71(d,J＝5.9Hz,1H),4.87–4.37(m,6H),2.14(s,6H). Two aliphatic protons CH ₂ overlap with H ₂ O. Formate. LCMS, M/z: [ m+h ] ⁺ = 543.23.

Example 6

(Method A)

¹H NMR(400MHz,DMSO-d6)δ9.70(s,1H),8.64(s,1H),8.41(d,J＝5.8Hz,1H),8.27(s,1H),8.21(dd,J＝8.4,1.3Hz,1H),7.82(d,J＝4.9Hz,4H),7.75(d,J＝8.4Hz,1H),7.70(d,J＝1.0Hz,1H),7.49(s,1H),7.12(s,1H),6.73(d,J＝5.8Hz,1H),5.02–4.41(m,10H),3.62–3.52(m,1H). Formate. LCMS, M/z: [ m+h ] ⁺ = 542.14.

Example 7

(Method A)

¹H NMR(300MHz,DMSO-d6)δ9.68(s,1H),8.54(s,1H),8.40(d,J＝5.9Hz,1H),8.24–8.14(m,2H),7.87–7.77(m,4H),7.74(d,J＝8.4Hz,1H),7.70(s,1H),7.53(s,1H),7.02(s,1H),6.72(d,J＝5.9Hz,1H),4.31–3.96(m,4H),3.95(s,3H),3.91–3.71(m,2H). Formate. LCMS, M/z: [ m+h ] ⁺ = 500.39.

Example 8

(Method A)

¹H NMR(400MHz,DMSO-d6)δ9.73(s,1H),9.10(d,J＝8.8Hz,1H),8.56(s,1H),8.41(d,J＝5.8Hz,1H),8.33(s,4H),8.20(dd,J＝8.4,1.3Hz,1H),7.87–7.75(m,5H),7.71(d,J＝1.0Hz,1H),7.50(s,1H),7.23(s,1H),6.73(d,J＝5.8Hz,1H),4.87(dt,J＝15.3,7.7Hz,1H),4.72(t,J＝6.9Hz,2H),2.60(t,J＝6.8Hz,2H),2.16(s,6H),1.40(d,J＝7.0Hz,3H). Formate (1:4). LCMS, M/z: [ m+h ] ⁺ = 563.23.

Example 9

(Method A)

¹H NMR(300MHz,DMSO-d6)δ9.70(s,1H),8.99(d,J＝8.9Hz,1H),8.56(s,1H),8.41(d,J＝5.8Hz,1H),8.31(s,2H),8.20(dd,J＝8.4,1.3Hz,1H),7.86–7.75(m,4H),7.70(d,J＝1.1Hz,1H),7.53(s,1H),7.30(s,1H),6.73(d,J＝5.9Hz,1H),4.99–4.80(m,1H),4.09(s,3H),1.40(d,J＝7.1Hz,3H). Formate. LCMS, M/z: [ m+h ] ⁺ = 506.18.

Example 10

(Method A)

¹H NMR(400MHz,DMSO-d6)δ12.12(s,1H),9.70(s,1H),9.06(d,J＝8.9Hz,1H),8.63(s,1H),8.42(d,J＝5.8Hz,1H),8.21(d,J＝8.7Hz,1H),7.88–7.76(m,5H),7.71(s,1H),7.52(s,1H),7.32(s,1H),6.73(d,J＝5.9Hz,1H),5.00(dt,J＝15.4,7.5Hz,2H),4.95–4.84(m,1H),4.61(dt,J＝7.9,6.1Hz,2H),4.46(t,J＝6.1Hz,2H),3.51(p,J＝7.0Hz,1H),1.40(d,J＝7.0Hz,3H).LCMS,m/z:[M+H]⁺＝562.51.

Example 11

(Method A)

¹H NMR(400MHz,DMSO-d6)δ12.11(s,1H),9.69(s,1H),8.61(s,1H),8.41(d,J＝5.8Hz,1H),8.20(dd,J＝8.4,1.3Hz,1H),7.82(s,4H),7.74(d,J＝8.5Hz,1H),7.71(d,J＝1.1Hz,1H),7.52(s,1H),7.04(s,1H),6.72(d,J＝5.8Hz,1H),4.81(d,J＝7.4Hz,2H),4.62(dd,J＝7.8,6.1Hz,2H),4.42(s,2H),3.98(dd,J＝84.9,48.3Hz,4H),3.47(s,2H). One proton CH overlaps H ₂ O. LCMS, M/z: [ m+h ] ⁺ = 556.14.

Example 12

(Method A)

¹H NMR(400MHz,DMSO-d6)δ9.68(s,1H),8.53(s,1H),8.40(d,J＝5.8Hz,1H),8.30(s,3H),8.18(dd,J＝8.4,1.4Hz,1H),7.84–7.77(m,4H),7.72(d,J＝8.4Hz,1H),7.70(d,J＝1.1Hz,1H),7.52(s,1H),6.90(s,1H),6.72(d,J＝5.9Hz,1H),3.93(s,3H),3.66(t,J＝6.3Hz,2H),3.55(t,J＝6.6Hz,2H),1.91(d,J＝8.3Hz,4H). Formate (1:3). LCMS, M/z: [ m+h ] ⁺ = 464.39.

Example 13

(Method A)

¹H NMR(400MHz,DMSO-d₆)δ9.70(s,1H),8.57(s,1H),8.42(d,J＝5.8Hz,1H),8.21(dd,J＝8.5,1.4Hz,1H),8.14(s,1H),7.87–7.73(m,5H),7.55(s,1H),7.06(s,1H),6.73(d,J＝5.9Hz,1H),4.66(s,2H),4.20(s,2H),4.00(s,2H),3.80(s,2H),3.05(s,2H). Six aliphatic protons 2xCH ₃ overlap DMSO-d 6. LCMS, M/z: [ m+h ] ⁺ = 557.20.

Example 14

(Method A)

¹H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.58(s,1H),8.44–8.39(m,2H),8.28(s,2H),7.91(d,J＝8.3Hz,1H),7.80(q,J＝8.6Hz,4H),7.70(d,J＝1.1Hz,1H),7.66(d,J＝1.0Hz,1H),7.55(s,1H),6.77(d,J＝5.9Hz,1H),5.12(s,2H),4.56(s,2H). Formate (1:2). LCMS, M/z: [ m+h ] ⁺ = 473.29.

Example 15

(Method B)

¹H NMR(300MHz,DMSO-d6)δ9.82(s,1H),8.56(s,1H),8.44(d,J＝5.8Hz,1H),8.28(s,1H),8.20(dd,J＝8.4,1.3Hz,1H),8.14(s,1H),7.97–7.87(m,4H),7.74(d,J＝8.4Hz,1H),7.01(s,1H),6.75(d,J＝5.8Hz,1H),4.64(t,J＝6.0Hz,6H),2.17(s,6H). Two aliphatic protons CH ₂ overlap DMSO. Formate. LCMS, M/z: [ m+h ] ⁺ = 544.38.

Example 16

(Method B)

¹H NMR(400MHz,DMSO-d6)δ9.84(s,1H),8.53(s,1H),8.44(s,1H),8.40(s,3H),8.30(s,1H),8.19(dd,J＝8.4,1.4Hz,1H),7.93(q,J＝8.7Hz,4H),7.73(d,J＝8.4Hz,1H),6.91(s,1H),6.76(d,J＝5.8Hz,1H),3.94(s,3H),3.67(s,2H),3.54(d,J＝6.9Hz,2H),1.90(s,4H). Formate (1:3). LCMS, M/z: [ m+h ] ⁺ = 465.37.

Example 17

(Method B)

¹H NMR(400MHz,DMSO-d6)δ9.84(s,1H),8.55(s,1H),8.44(d,J＝5.8Hz,1H),8.35(s,1H),8.30(s,1H),8.20(dd,J＝8.4,1.4Hz,1H),7.93(q,J＝8.8Hz,4H),7.75(d,J＝8.5Hz,1H),7.02(s,1H),6.76(d,J＝5.8Hz,1H),4.20(s,2H),3.95(s,4H),3.81(s,1H). Two aliphatic protons CH ₂ overlap with H ₂ O. Formate. LCMS, M/z: [ m+h ] ⁺ = 501.20.

Example 18

(Method B)

¹H NMR(400MHz,DMSO-d₆)δ9.81(s,1H),8.55(s,1H),8.44(d,J＝5.8Hz,1H),8.26(s,1H),8.19(dd,J＝8.4,1.3Hz,1H),8.14(s,1H),7.92(q,J＝8.7Hz,4H),7.74(d,J＝8.4Hz,1H),6.97(d,J＝3.2Hz,1H),6.75(d,J＝5.8Hz,1H),4.57(s,2H),4.14(s,2H),3.96(s,2H),3.78(s,2H),2.19(s,6H). Two aliphatic protons CH ₂ overlap with DMSO-d 6. Formate. LCMS, M/z: [ m+h ] ⁺ = 558.16.

Example 19

(Method B)

¹H NMR(400MHz,DMSO-d6)δ9.91(s,1H),8.58(s,1H),8.46(d,J＝5.8Hz,1H),8.42(dd,J＝8.2,1.4Hz,1H),8.39(s,1H),8.32(s,1H),7.96–7.88(m,5H),7.67(d,J＝1.0Hz,1H),6.81(d,J＝5.9Hz,1H),5.12(s,2H),4.56(s,2H). Formate. LCMS, M/z: [ m+h ] ⁺ = 474.20.

Example 20

(Method B)

¹H NMR(400MHz,DMSO-d6)δ12.14(s,1H),9.74(s,1H),8.98(d,J＝1.4Hz,1H),8.46(dd,J＝8.5,1.5Hz,1H),8.43(d,J＝5.9Hz,1H),8.08(d,J＝8.5Hz,1H),8.01(s,1H),7.79(s,4H),7.71(d,J＝1.1Hz,1H),7.59(d,J＝20.2Hz,1H),6.77(d,J＝5.9Hz,1H),4.85(d,J＝199.8Hz,4H). A residue containing formic acid. LCMS, M/z: [ m+h ] ⁺ = 489.10.

Example 21

(Method B)

¹H NMR(400MHz,DMSO-d6)δ13.60(d,J＝36.6Hz,1H),9.72(s,1H),8.68(d,J＝34.4Hz,1H),8.50–8.33(m,2H),8.20(d,J＝3.9Hz,2H),7.81(s,5H),7.71(d,J＝1.1Hz,1H),7.53(s,1H),6.73(d,J＝5.8Hz,1H),5.16(d,J＝13.0Hz,2H),4.62(t,J＝12.3Hz,2H). Formate. LCMS, M/z: [ m+h ] ⁺ = 472.88.

Example 22

(Method B)

¹H NMR(400MHz,DMSO-d6)δ13.65(s,1H),9.85(s,1H),8.67(s,1H),8.44(d,J＝5.8Hz,1H),8.37(s,2H),8.30(s,1H),7.93(s,4H),7.81(d,J＝12.2Hz,1H),6.77(d,J＝5.8Hz,1H),5.18(t,J＝12.3Hz,2H),4.62(t,J＝12.2Hz,2H).LCMS,m/z:[M+H]⁺＝474.25.

Example 23

(Method C)

¹H NMR(400MHz,DMSO-d₆)δ9.34(s,1H),8.17(s,2H),7.97(d,J＝5.7Hz,1H),7.76–7.67(m,3H),7.63(d,J＝8.4Hz,2H),7.50(s,1H),7.38(s,1H),6.10(d,J＝5.7Hz,1H),4.96(s,2H),4.12(s,1H),4.04(t,J＝5.8Hz,4H),3.77(s,2H),2.75(s,2H). Formate (1:2). LCMS, M/z: [ m+h ] ⁺ = 520.11.

Example 24

(Method C)

¹H NMR(400MHz,DMSO-d6)δ9.49(s,1H),8.32(s,2H),8.27(s,1H),8.00(d,J＝5.7Hz,1H),7.88–7.80(m,2H),7.75(d,J＝8.7Hz,2H),7.38(s,1H),6.13(d,J＝5.7Hz,1H),4.97(s,2H),4.12(s,1H),4.05(t,J＝5.9Hz,4H),3.77(s,2H),2.76(s,3H),2.61(s,1H). Formate (1:2). LCMS, M/z: [ m+h ] ⁺ = 521.09.

Example 25

(Method C)

¹H NMR(400MHz,DMSO-d6)δ12.12(s,1H),i9.34(s,1H),8.28(s,2H),7.97(d,J＝5.6Hz,1H),7.73(s,2H),7.68(d,J＝1.1Hz,1H),7.63(d,J＝7.9Hz,2H),7.54(s,1H),7.42(s,1H),6.10(d,J＝5.7Hz,1H),4.97(s,2H),4.21(bs,1H),4.04(t,J＝5.8Hz,3H),3.96–3.69(m,4H),2.76(s,2H). Formate (1:2). LCMS, M/z: [ m+h ] ⁺ = 508.42.

Comparative example

The structure of comparative compound a is provided below:

it can be prepared according to the method found in WO 2019/001572.

Example 26

ADP-Glo kinase assay (Promega). The ADP-Glo ^TM kinase assay is a luminescent ADP detection assay that allows for the measurement of kinase activity based on the amount of ADP produced during the kinase reaction. The kinase reaction is performed in the presence of ATP, S6K substrate (KRRRLASLR) and ROCK kinase in an appropriate kinase reaction buffer (1X). At the end of the reaction, unconsumed ATP is depleted by the addition of ADP-Glo ^TM reagent. Next, kinase detection reagents are added to convert ADP to ATP and the newly synthesized ATP is measured using the luciferase/luciferin reaction. Luminescence is proportional to ADP produced and thus to kinase activity. ADP-Glo ^TM kinase assays were performed in 384 well plates as follows.

Kinase assay reagents were prepared by thawing the kinase assay buffer at Room Temperature (RT). If a pellet is present, the buffer is incubated at 37 ℃ for 15 minutes under constant vortex to dissolve the pellet or remove the pellet from the buffer by carefully pipetting the supernatant from the bottle. After equilibration of both the kinase assay buffer and the kinase assay substrate to room temperature, the entire volume of kinase assay buffer was transferred to a bottle containing the kinase assay substrate to reconstitute the lyophilized substrate, followed by gentle mixing to obtain a homogeneous solution.

Kinase reaction buffer preparation kinase reaction buffer was freshly prepared prior to each experiment. The formulation of this buffer is provided in table 1 below:

table 1 preparation of kinase reaction buffer (1X) for ADP-Glo kinase assay.

Reagent:	Final concentration	Concentration of stock solution
			Tris,pH 7.5	40mM	500MM aqueous solution
DTT	0.05mM	100MM aqueous solution
			MgCl₂	20mM	500MM aqueous solution
BSA	0.1mg/mL	5.0mg/mL

ADP-Glo reagent preparation. Three mixtures were prepared on ice before performing the assay:

mixture 1-containing ROCK1 or ROCK2 (1X) in kinase reaction buffer

Mixture 2-containing S6K peptide (1 x) in kinase reaction buffer

Mixture 3-containing ATP (1 x) in kinase reaction buffer

The final conditions for performing the ADP-Glo kinase assay are shown in Table 2 below:

Table 2. Final conditions for ADP-Glo kinase assay of ROCK (concentration in final volume 5. Mu.L).

Final concentration:	ROCK1	ROCK2
			Kinase enzymes	0.1ng/μL	0.1ng/μL
S6K substrate	170μM	170μM
			ATP	10μM	40μM
Time point	120 Minutes	120 Minutes
			DMSO	1%	1%

The volume ratio of the reagent in the measurement is 1:1:2, and the reagent is ADP-Glo ^TM and the reagent is kinase detection reagent. For 384 well plates, the volumes were 5. Mu.l kinase reactant, 5. Mu.l ADP-Glo ^TM reagent and 10. Mu.l kinase detection reagent.

Standard curves were generated for ATP conversion to ADP. To evaluate the amount of ADP produced in the kinase reaction, a standard curve representing luminescence corresponding to the conversion of ATP to ADP was prepared based on the ATP concentration used in the kinase reaction. These conversion curves represent the amounts of ATP and ADP available in the reaction at the indicated conversion percentages ranging from 100% conversion to 0% conversion. Standard samples for generating ATP-ADP conversion curves were generated by combining appropriate volumes of ATP and ADP stock solutions.

Determination of IC ₅₀ value for kinase inhibitors

Each plate contained positive controls (1. Mu.M Compound A for ROCK2 and 1. Mu. MRKI-1447 for ROCK 1), vehicle controls (1% DMSO), blank controls (kinase reaction buffer (1X)), low controls (substrate and ATP, no kinase), and autophosphorylated controls (kinase and ATP, no substrate). Test and reference compounds were diluted in 100% DMSO to obtain 10mM stock solutions. Each compound was tested in duplicate in 8-fold serial dilutions. The final concentration of DMSO in the reaction was 1% (up to 50 nL).

Mu.l/well kinase reaction buffer (1X) was dispensed to the blank wells. Mu.l/well kinase reaction buffer (1X) was dispensed into control wells containing no protein (low control) and no substrate (autophosphorylation). The plates were sealed with an adhesive film and then briefly spun (1 minute, 1000 rpm).

Mu.l/Kong Jimei (2.5X) of the solution was dispensed into the relevant wells of the assay plate. The plates were sealed with an adhesive film and then briefly spun (1 minute, 1000 rpm).

Mu.l/well of S6K substrate (2.5X) solution was dispensed into the relevant wells of the assay plate. The plates were sealed with an adhesive film and then briefly spun (1 minute, 1000 rpm).

Mu.l/well of ATP (5X) solution was dispensed into the relevant wells of the assay plate. The final volume of the reaction was 15. Mu.l. The plates were sealed with an adhesive film and then briefly spun (1 minute, 1000 rpm).

Plates were incubated at 25℃for 120 minutes at a shaking speed of 450 rpm.

After the incubation is complete, the kinase reaction is terminated, residual ATP is depleted from the kinase reaction and ADP is converted to ATP, which is measured in the luciferase/luciferin reaction. The 5 μ lADP-Glo ^TM reagent was dispensed to stop the kinase reaction and deplete unconsumed ATP. The plates were sealed with an adhesive film, then spun briefly (1 min, 1000 rpm) and incubated at 25 ℃ for 40 min with shaking at 450 rpm.

Mu.l of kinase assay reagent was dispensed to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP. The plates were sealed with an adhesive film, then spun briefly (1 min, 1000 rpm) and incubated at 25 ℃ for 60 min with shaking at 450 rpm.

After 5 minutes dark adaptation, luminescence was measured using PHERASTAR FSX multimode plate reader (BMG Labtech).

IC ₅₀ parameters Log (inhibitor) and response-variable slope were calculated using a 4 parameter model using GRAPHPAD PRISM 7.04.04 software after normalization.

Example 28

Selective ROCK2 inhibition down-regulates IL-17 secretion via pSTAT 3-dependent mechanisms

Rho kinase (ROCK) is a member of the serine/threonine kinase family, often studied for its role in cell morphology, motility and shape by its effect on the cytoskeleton. Although both isoforms of ROCK, ROCK1 and ROCK2, share more than 90% homology within their kinase domains, the function of these proteins is not redundant and depends on the cellular system in which ROCK is expressed and activated. Recent studies have shown that ROCK2, but not ROCK1, regulates the pro-inflammatory IL-17 production lineage of T cells called Th17 cells via STAT 3-dependent mechanisms. Deregulated activation of Th17 cells and STAT3 phosphorylation have been implicated in the pathogenesis of inflammatory hyperlipidemia as well as fibrotic pathologies. Furthermore, selective ROCK2 inhibition alters the balance between pro-inflammatory Th17 and immunosuppressive regulatory T cells (tregs) by increasing STAT5 phosphorylation and IL-10 secretion.

By using a combination of stimulatory antibodies against CD3 and CD28 (anti-CD 3/CD 28) with IL-1β and TGF- β (Th 17-shift conditions), we found novel selective ROCK2 inhibitors, such as the compounds of example 1 and example 2, robustly down-regulate IL-17 secretion in Th 17-shifted human CD4+ T cells in a dose-dependent manner with EC50 at 838nM and 764nM, respectively (see FIGS. 2A and 2B). In this study, peripheral blood CD4 ⁺ T cells were treated with the indicated dose of ROCK2 inhibitor and then stimulated with anti-CD 3/28mAb, IL-1β (50 ng/mL) and TGF- β (5 ng/mL) for 48 hours. The supernatant was analyzed for IL-17 by ELISA. A representation of three different experiments is shown.

Induction and expression of IL-17 and other pro-inflammatory cytokines has been shown to depend on activation and phosphorylation of certain transcription factors, including STAT 3. Treatment of human CD4 ⁺ T cells with selective ROCK2 inhibitors during Th 17-shift conditions resulted in a dose-dependent decrease in STAT3 phosphorylation (fig. 3), consistent with the ability of the compounds of example 1 and example 2 to down-regulate IL-17 secretion and pCofilin (classical downstream ROCK target) under the same stimulation conditions (fig. 4). In these studies, peripheral blood CD4 ⁺ T cells were treated with the indicated doses of inhibitor and then stimulated with anti-CD 3/28mAb, IL-1β (50 ng/mL) and TGF- β (5 ng/mL) for 2 hours. Cell lysates were prepared and analyzed by western blot. A representation of three different experiments is shown.

Example 27

Selective ROCK2 inhibitors down-regulate the expression of pro-fibrogenic genes in fibroblasts

ROCK is activated by small gtpase Rho in response to multiple pro-fibrotic signals and regulates cytoskeletal dynamics, activation of diverse downstream intracellular targets, and expression of key pro-fibrotic genes such as fibronectin, smooth muscle actin (α -SMA), and collagen 3 (Col 3 A1). By using MRC-5 human fibroblasts pretreated with different doses of selective ROCK2 inhibitor, we found that both the compounds of example 1 and example 2 down-regulated TGF- β induced fibronectin as determined by PCR, α -SMA and Col3A1 gene expression (fig. 5) and Col1A protein production in supernatants as determined by ELISA (fig. 6). These data further demonstrate that selective ROCK2 inhibition has a direct robust anti-fibrosis effect.

In these studies, human lung fibroblast MRC-5 cells were obtained from the American Type Culture Collection (ATCC) and cultured in MEM supplemented with 10% fetal bovine serum. Cells were seeded at 50,000/mL/well in 24-well cell culture plates. The following day, cells were starved overnight in MEM containing 0.5% FBS. TGF-. Beta.1 (2.5 ng/mL) and various concentrations of the compounds of example 1 and example 2 were applied to cells for 48 hours. Cellular mRNA was isolated and analyzed by RT-PCR for mRNA expression of the indicated genes (fibronectin, αSMA, and col 3A) (FIG. 5). Secreted procollagen 1a (Col 1 A1) in the supernatant was measured by ELISA (fig. 6).

Example 28

Selective ROCK2 inhibitors effectively down-regulate differentiation in human adipocytes

Adipogenesis is a multi-step, complex process that progresses from precursor stem cells to adipocytes that synthesize and store fat. Dysregulation of adipocyte function has been shown to be involved in the pathogenesis of a wide range of metabolic diseases such as type 2 diabetes, obesity, etc. ROCK has been suggested to be involved in the regulation of adipocyte differentiation, however the isoform-specific contribution to adipogenesis and other metabolic pathways is not well characterized. Recent data indicate that KD025, a selective ROCK2 inhibitor, down regulates adipocyte differentiation in both mouse 3T3-L1 preadipocytes and human adipose derived stem cells by reducing expression of key adipogenic/lipogenic genes such as PPARg, C/EBPa, and Glut 4. By using primary human adipocytes, we found that the novel selective ROCK2 inhibitors, examples 1 and 2, down-regulate adipogenesis in a dose-dependent manner (fig. 7), further confirming the antimetabolite potential of ROCK2 targeting in cells. In this study, human subcutaneous adipocytes were grown to confluence, after which differentiation was induced by adipogenic mixture (0.1. Mu.M dexamethasone, 1. Mu.M insulin, 200. Mu.M indomethacin, 250. Mu.M Isobutylmethylxanthine (IBMX)) in the presence of the indicated concentrations of example 1 and example 2 for 10 days. Differentiated cells were stained with oil red, extracted with isopropanol and measured by absorbance at 492 nM.

Claims

1. A compound having formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, halo, -CN, C ₁ -C ₃ perfluoroalkyl, -OR ¹¹ , -O-(C ₁ -C ₆ alkyl)-OR ¹¹ , -(C ₁ -C ₆ alkyl)-OR ¹¹ , -NR ¹¹ R ¹² , -O-(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl)-OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -O-(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ , -C(═O)-R ¹¹ , -C(═O)OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ and NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ ;

^R2 is selected from the group consisting of H, _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, C3 _-C7 _cycloalkyl , halo, -CN, _C1 - _C3 perfluoroalkyl, ^-OR11 , -O-( _C1 - _C6 alkyl) ^-OR11 , -(C1-C6 ^alkyl ) ^-OR11 , ^-NR11R12 , -O-( _C1 - _C6 alkyl)-NR11R12, -(C1 _- _C6 alkyl) ^-NR11R12 ^, ^-NR11- ( _C1 - _C6 alkyl ₎ ^-NR11R12 , ^-NR11- ( _C1 - _C6 _alkyl ) ^-OR11 ^, -( _C1 - ^C6 _alkyl ⁾ _x -C(═O) ^R11 , -O-(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ , -C(═O)-R ¹¹ , -C(═O)OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ and -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ ;

Alternatively, R ¹ and R ² together form a 5- or 6-membered saturated or unsaturated fused ring, the fused ring can contain 0 to 2 ring heteroatoms selected from the group consisting of N, O and S, and the fused ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of: C ₁ -C ₆ alkyl, halo, -CN, -OH, oxo, -O-(C ₁ -C ₆ alkyl), -O-(C ₁ -C ₆ alkyl)-OH, -O-(C ₁ -C ₆ alkyl)-O-(C ₁ -C ₆ alkyl), -NR ¹¹ R ¹² , -O-(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , C ₁ -C ₃ perfluoroalkyl, -NR ¹¹ -(C ₁ -C ₆ alkyl)NR ¹¹ R ¹² and -NR ¹¹ -(C ₁ -C ₆ alkyl)-OR ¹¹ ;

^X4 is N or CH;

^R3 and ^R4 are each independently selected from the group consisting of H, _C1 - _C6 alkyl, C2 _-C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C7 cycloalkyl, 3- to ₁₀ -membered heterocyclyl, _C6 - _C10 aryl, 5- to 14-membered heteroaryl, _C6-12 aralkyl, -( _C1 - _C6 alkyl) ^-OR11 , -(C1- _C6 alkyl) ^-NR11R12 , -( _C1 - _C6 alkyl) _x -C(═O) ^R11 , -( _C1 - _C6 alkyl) _x -C( ^═O ) ^OR11 , and -( _C1 _- _C6 alkyl) _x - ^C (═O) ^NR11R12 ;

Alternatively, ^R3 and ^R4, together with the nitrogen to which they are attached, provide (i) a 4- to 6-membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from N, O, and S, or (ii) a 5- to 10-membered heterobicyclic ring system having 0 to 3 additional ring heteroatoms selected from N, O, and S; wherein the heterocyclic ring or the heterobicyclic ring system is unsubstituted or substituted with 1 to 4 substituents selected from the group consisting of halo, _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C7 cycloalkyl, halo, -CN, _C1 - _C3 perfluoroalkyl, ^-OR11 , oxo, -O-( _C1 - _C6 alkyl) ^-OR11 , -( _C1 - _C6 alkyl) ^-OR11 , ^-NR11R12 , -O-( ^C1 _- _C6 alkyl) ^-NR11R -(C ₁ -C ₆ alkyl)x ^- C( ^═O) R ¹¹ , ^-O- (C ₁ -C ₆ alkyl)x-C( ^═O ^{)R 11} ^, -(C ₁ _-C ₆ alkyl) _x -C( _═O )OR ¹¹ , -C( _═O )-R ¹¹ , _-C (═O)OR ¹¹ , -(C ₁ _-C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ^11- (C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ ^, and -NR ^11- (C ₁ -C ⁶ alkyl) _x - _C ₍ ═O)R ¹¹ ₆ alkyl) _x -C(═O)OR ¹¹ ;

Dashed lines represent optional double bonds;

Each R ⁵ is independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, halo, -CN, C ₁ -C ₃ perfluoroalkyl, oxo, -OR ¹¹ , -O-(C ₁ -C ₆ alkyl)-OR ¹¹ , -(C ₁ -C ₆ alkyl)-OR ¹¹ , -NR ¹¹ R ¹² , -O-(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl)-OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)R ^-O- (C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ , -C(═O)-R ¹¹ , -C(═O)OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ and -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ ;

n is 0 to 3;

^X1 is selected from the group consisting of ^CR6 and N;

X ² is selected from the group consisting of CHR ⁶ , NR ⁷ , O and S;

X ³ is selected from the group consisting of C, CH and N;

Each R ⁶ is independently selected from the group consisting of H, C ₁ -C ₆ , alkyl, C ₂ -C ₆ , alkenyl, C ₂ -C ₆ , alkynyl, C ₃ -C ₇ , cycloalkyl, halo, -CN, C ₁ -C ₃ , perfluoroalkyl, -OR ¹¹ , -O-(C ₁ -C ₆ , alkyl)-OR ¹¹ , -(C ₁ -C ₆ , alkyl)-OR ¹¹ , -NR ¹¹ R ¹² , -O-(C ₁ -C ₆ , alkyl)-NR ¹¹ R ¹² , -(C ₁ -C ₆ , alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ , alkyl)-NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ , alkyl)-OR ¹¹ , -(C ₁ -C ₆ , alkyl) _x -C(═O)R ¹¹ , -O-(C ₁ -C ₆ , alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ , alkyl) _x -C(═O)OR ¹¹ , -C(═O)-R ¹¹ , -C(═O)OR ¹¹ , -(C ₁ -C ₆ , alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ , alkyl) _x -C(═O)R ¹¹ and -NR ¹¹ -(C ₁ -C ₆ , alkyl) _x -C(═O)OR ¹¹ ;

each R ⁷ is independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ¹¹ , -(C ₁ -C ₆ alkyl)-NR ¹¹ R ¹² , -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ , and -(C ₁ -C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² ;

Each x is independently selected from 0 and 1; and

Each R ¹¹ and R ¹² is independently selected from the group consisting of H and C ₁ -C ₆ alkyl;

Or alternatively, when R ¹¹ and R ¹² are both attached to the same nitrogen, they together form a 4- to 7-membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from the group consisting of N, O and S, and said heterocyclic ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, -CN, -NH ₂ , C ₁ -C ₃ perfluoroalkyl, -OH, -O-(C ₁ -C ₆ alkyl) and -(C ₁ -C ₆ alkyl)-OH.

2. The compound according to claim 1, which has formula II:

or a pharmaceutically acceptable salt thereof, wherein:

^R2 _{is selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7} _cycloalkyl _, _halo _, _-CN _, _C1 _- _C3 perfluoroalkyl, ^-OR11 , -O-( _C1 - _C6 alkyl) ^-OR11 , -(C1-C6 ^alkyl ) ^-OR11 , ^-NR11R12 , -O-( _C1 - _C6 alkyl)-NR11R12, -(C1 _- _C6 alkyl) ^-NR11R12 ^, ^-NR11- ( _C1 - _C6 alkyl ₎ ^-NR11R12 , ^-NR11- ( _C1 - _C6 _alkyl ) ^-OR11 ^, -( _C1 - ^C6 _alkyl ⁾ _x -C(═O) ^R11 , -O-(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ , -C(═O)-R ¹¹ , -C(═O)OR ¹¹ , -(C ₁ -C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ and -NR ¹¹ -(C ₁ -C ₆ alkyl) _x -C(═O)OR ¹¹ ;

Alternatively, ^R3 and ^R4, together with the nitrogen to which they are attached, provide (i) a 4- to 6-membered heterocyclic ring having 0 to 2 additional ring heteroatoms selected from N, O, and S, or (ii) a 5- to 10-membered heterobicyclic ring system having 0 to 3 additional ring heteroatoms selected from N, O, and S; wherein the heterocyclic ring or the heterobicyclic ring system is unsubstituted or substituted with 1 to 4 substituents selected from the group consisting of halo, _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C7 cycloalkyl, halo, -CN, _C1 - _C3 perfluoroalkyl, ^-OR11 , oxo, -O-( _C1 - _C6 alkyl) ^-OR11 , -( _C1 - _C6 alkyl) ^-OR11 , ^-NR11R12 , -O-( ^C1 _- _C6 alkyl) ^-NR11R -(C ₁ -C ₆ alkyl)x-C( ^═O ⁾ R ¹¹ , ^-O- (C ₁ -C ₆ alkyl)x-C( ^═O ^{)R 11} ^, -(C ₁ _-C ₆ alkyl) _x -C( _═O )OR ¹¹ , -C( _═O )-R ¹¹ , _-C (═O)OR ¹¹ , -(C ₁ _-C ₆ alkyl) _x -C(═O)NR ¹¹ R ¹² , -NR ^11- (C ₁ -C ₆ alkyl) _x -C(═O)R ¹¹ ^, and -NR ^11- (C ₁ -C ⁶ alkyl) _x - _C ₍ ═O)R ¹¹ ₆ alkyl) _x -C(═O)OR ¹¹ ;

Dashed lines represent optional double bonds;

n is 0 to 3;

^X1 is selected from the group consisting of ^CR6 and N;

X ² is selected from the group consisting of CHR ⁶ , NR ⁷ , O and S;

X ³ is selected from the group consisting of C, CH and N;

Each x is independently selected from 0 and 1; and

3. The compound according to claim 1, which has the formula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

Dashed lines represent optional double bonds;

^X1 is selected from the group consisting of ^CR6 and N;

X ² is selected from the group consisting of CHR ⁶ , NR ⁷ , O and S;

X ³ is selected from the group consisting of C, CH and N;

Each x is independently selected from 0 and 1; and

4. The compound according to claim 1, which has the formula III:

or a pharmaceutically acceptable salt thereof, wherein:

Dashed lines represent optional double bonds;

n is 0 to 3;

^X1 is selected from the group consisting of ^CR6 and N;

X ² is selected from the group consisting of CHR ⁶ , NR ⁷ , O and S;

X ³ is selected from the group consisting of C, CH and N;

Each x is independently selected from 0 and 1; and

5. The compound according to claim 1, which has the formula IIIa:

or a pharmaceutically acceptable salt thereof, wherein:

Dashed lines represent optional double bonds;

^X1 is selected from the group consisting of ^CR6 and N;

X ² is selected from the group consisting of CHR ⁶ , NR ⁷ , O and S;

X ³ is selected from the group consisting of C, CH and N;

Each x is independently selected from 0 and 1; and

6. The compound according to claim 1, which is 2-[2-(3,3-difluoroazetidine-1-carbonyl)-1-methyl-1H-indol-6-yl]-N-[4-(1H-imidazol-5-yl)phenyl]pyrimidin-4-amine having the following formula:

or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1, which is 2-[2-(3,3-difluoroazetidine-1-carbonyl)-1-methyl-1H-indol-6-yl]-N-[4-(1H-1,2,3-triazol-4-yl)phenyl]pyrimidin-4-amine having the following formula:

or a pharmaceutically acceptable salt thereof.

8. A method for treating a disease or condition mediated by ROCK2, wherein the method comprises administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the disease or disorder is selected from the group consisting of fibrotic diseases, inflammatory diseases, autoimmune diseases, cardiovascular disorders, central nervous system disorders, neoplastic diseases, metabolic syndrome, eye diseases, kidney diseases, lung diseases, muscular dystrophy, sickle cell disease, and viral diseases.

10. The method of claim 9, wherein the disease or condition is selected from the group consisting of a fibrotic disease, an inflammatory disease, and an autoimmune disease.

11. The method of claim 10, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus (SLE; lupus), psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, atopic dermatitis, eczema, or graft-versus-host disease (GVHD; acute and chronic), idiopathic pulmonary fibrosis, and scleroderma.

12. The method of claim 9, wherein the disease or disorder is selected from the group consisting of a cardiovascular disorder, a central nervous system disorder, a neoplastic disease, or a metabolic syndrome.

13. The method of claim 10, wherein the inflammatory disorder is selected from the group consisting of cardiovascular inflammation, lung inflammation, kidney inflammation, arteriosclerosis, and sepsis.

14. The method of claim 10, wherein the fibrotic disorder is selected from the group consisting of idiopathic pulmonary fibrosis, renal fibrosis, kidney fibrosis, ocular fibrosis, cardiac fibrosis, NASH, scleroderma, systemic sclerosis, and liver cirrhosis.

15. The method of claim 12, wherein the neoplastic disease is selected from the group consisting of ovarian cancer, breast cancer, and pancreatic cancer.

16. The method of claim 12, wherein the cardiovascular disease is selected from the group consisting of hypertension, cardiomyopathy, cardiac remodeling, atherosclerosis, restenosis, cardiac hypertrophy, cerebral ischemia, cerebral vasospasm, and erectile dysfunction.

17. The method of claim 9, wherein the pulmonary disease is selected from the group consisting of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and asthma.

18. The method of claim 9, wherein the central nervous system disorder is selected from the group consisting of neuronal degeneration or spinal cord injury, traumatic brain injury, cerebral cavernous malformation, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

19. The method of claim 9, wherein the kidney disease is selected from the group consisting of polycystic kidney disease, renal fibrosis, and diabetic kidney disease.

20. The method of claim 9, wherein the metabolic disease is selected from the group consisting of insulin resistance, hyperinsulinemia, type 2 diabetes, obesity, metabolic syndrome, and glucose intolerance.

21. The method of claim 9, wherein the ocular disease is selected from the group consisting of ocular hypertension, age-related macular degeneration (AMD; wet and dry), choroidal neovascularization (CNV), choroidal tumors, diabetic macular edema (DME), iris neovascularization, uveitis, glaucoma, primary open-angle glaucoma, acute angle-closure glaucoma, pigmentary glaucoma, congenital glaucoma, normal-tension glaucoma, secondary glaucoma, neovascular glaucoma, geographic atrophy, and retinitis of prematurity (ROP).

22. The method of claim 9, wherein the disease is Duchenne muscular dystrophy.

23. The method of claim 9, wherein the viral infection is a coronavirus infection, such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV.